Atherosclerosis is a chronic disease of arterial blood vessels and is fundamentally linked to abnormally high blood cholesterol levels. Atherosclerotic lesions develop after excessive amounts of lipids accumulate in arterial walls. Macrophages in atherosclerotic lesions take up and store large amounts of lipids during which they are activated to produce inflammatory cytokines and growth factors. The local saturation of free cholesterol can lead to crystal formation inside macrophages and in the extracellular space. Inflammatory immune responses in atherosclerotic lesions are key pathogenic factors for the development of clinical disease. However, the factors leading to the activation of inflammation in atherosclerotic lesions remain unknown. Immune cells recognize and respond to conditions that are of danger to the host, such as infections, cancer, tissue damage or metabolic derangements. Immune cells express receptors that can recognize foreign molecules from microbes as well as altered self-molecules, which appear under pathologic conditions. It has recently been shown that crystallization of normally soluble material is perceived as a danger to the host and can lead to the activation of the cytosolic receptor complex NALP3 inflammasome. We found that cholesterol crystals, which are abundantly present in atherosclerotic lesions, could activate the NALP3 inflammasome in immune cells leading to caspase-1 mediated maturation and release of the pro-inflammatory cytokines IL-12 and IL-18. Our hypothesis is that cholesterol crystals act as an endogenous danger signal and activate the NALP3 inflammasome. We postulate that cholesterol crystal-induced immune activation is fundamental for the development of inflammation in atherosclerotic lesions and could influence the initiation and progression of atherosclerotic disease. We propose to study the molecular mechanisms of cholesterol crystal recognition by analyzing mice with targeted lesions in components of the inflammasome pathway. Macrophages from knock-out mice will be analyzed in vitro and cholesterol crystal-induced inflammation will be tested in vivo. To better understand how crystals can enter macrophages and where signaling is initiated, we will make use of confocal imaging techniques that image crystals together with fluorescent structures at high spatial resolution. We will test the biological relevance of NALP3 inflammasome activation using a mouse atherosclerosis model with double knock-out mice that lack the LDL receptor and components of the inflammasome (NALP3 and ASC) pathway. We will further assess the prevalence of crystals in different human artherosclerotic lesion types together with cellular and inflamasome activation markers to gain insights into the relevance of crystals in human atherosclerotic disease. Detailed knowledge of the molecular mechanisms of cholesterol crystal recognition by immune cells will aid in the identification of novel therapeutic targets directed against the onset and progression of atherosclerosis, which could have broad clinical and socio-economic impact. PUBLIC HEALTH RELEVANCE: This project will lead to a detailed understanding of how immune cells are activated by cholesterol crystals. Understanding the molecular mechanisms of cholesterol crystal recognition can lead to novel molecular targets for therapies that prevent inflammation in atherosclerotic lesions, which can improve or prevent atherosclerotic disease.